The present invention relates to a recombinant vector suitable for use in gene therapy for insulin-dependent diabetes mellitus and a pharmaceutical composition comprising the recombinant vector as an effective ingredient. More particularly, the present invention relates to a gene therapy system for effectively and safely treating insulin-dependent diabetes mellitus by taking advantage of the gene delivery capacity of Drosophila""s P-transposon and the tissue specificity and expression enhancement of a K14 promoter.
Gene therapy offers a new paradigm for curing human diseases. Rather than altering disease phenotypes by using agents that interact with gene products or are themselves gene products, gene therapy theoretically can modify specific genes, which results in a cure following a single administration. Initially, gene therapy was envisioned for the treatment of genetic disorders, but is currently studied for a broad spectrum of diseases, including cancer, peripheral vascular disease, arthritis, neuro-degenerative disorders and other acquired diseases. Further, in combination with the Human Genome Project, gene therapy is expected to make a great progress in the treatment of far more diseases. With gene therapy, the delivery of genes into cells and their expression therein can be artificially regulated, so that the mutated genes of patients can be corrected by genetic recombination.
There are disclosed patents regarding gene therapy. For instance, PCT publication No. 1997-27310 claims a retrovirus vector which can be used in gene therapy and PCT publication No. 1997-34009 discloses a recombinant adenovirus vector for gene therapy for human tumors. Virus vectors are, however, limited to only the treatment of hereditary diseases, owing to safety concerns and highly complex procedures. Also, the gene therapy utilizing virus vectors, as in such patents, suffers from the disadvantage of requiring much time and high expense. In prior arts, non-viral insulin vectors have been disclosed nowhere yet.
Characterized by a grossly abnormal pattern of fuel usage-overproduction of glucose by the liver and under utilization by other organs, diabetes mellitus is a metabolic disease caused by insulin deficiency. Of various diabetes mellitus patterns, Insulin-dependent diabetes mellitus (IDDM), called Type I diabetes, results from the autoimmune destruction of the insulin-producing xcex2-cells of the pancreas. Currently, IDDM accounts for 3% of all new cases of diabetes each year with one incidence per 7,000 children.
Measures in current use for the treatment of IDDM include the monitoring of blood sugar levels, multiple injections of insulin, specialized diet, and exercise. In spite of faithful compliance with such intensive diabetes management strategies, patients can expect only a 50-70% reduction in the aggravation of diabetes. Therefore, there remains a need for developing better therapies.
The epidermis and its appendages, which are self-renewing tissues, have compartments of stem cells, each having the capacity to proliferate sufficiently to cover the body surface. The pioneering studies of Green et al. (1983) established that human skin keratinocytes could be serially propagated in culture for several hundred generations, resulting in the development of burn grafting operations employing cultured skin keratinocytes, which are now widely used in trauma units of major hospitals. Gene transfer into cultured keratinocytes has been demonstrated by utilizing a variety of different foreign promoters able to drive the expression of various secreted products. Keratinocytes within the epidermis are renewed by replicating cells which fall within the following two categories: (1) stem cells capable of extended or unlimited growth; and (2) transient amplifying cells, descended from stem cells, that replicate a limited number of times before undergoing terminal differentiation. Stem cells show slow cell cycles and are labeled infrequently with nucleotide analogues, but once labeled, retain that label for prolonged time periods. Stem cells and transient amplifying cells are located in compartments in the basal layer of the epidermis with terminally differentiated cells forming the stratified, super-basal layers. A stem cell and its descendant amplifying and terminally differentiated cells are clustered in a distinct spatial array termed the xe2x80x98epidermal proliferation unitxe2x80x99. Keratin 14 (K14) and its partner K5 are the major proteins expressed by active cells of the epidermis and its appendages, and the genes encoding these keratins are abundantly transcribed in cultured human keratinocytes. For these reasons, the K14 and K5 promoters are especially attractive candidates for use in keratinocyte-mediated gene therapy.
Since the first finding in the 1970s that P-transposon is included in the hybrid dysgenesis, Drosophila""s P-element has been under extensive study. A technique was reported in which cloned genes can be transferred to Drosophila""s embryos by use of P-transposon (Rubin, G. M., et al., Science, 1982, 218:348-353). However, this technique is not applicable even to allied species. Furthermore, the introduction of Drosophila""s transposon to mammals, as in the present invention, has not yet been reported thus far.
Leading to the present invention, the intensive and thorough research on gene therapy, conducted by the present inventors aiming to develop more effective, safer and simpler gene therapy for type I diabetes mellitus, resulted in the finding that a K14 promoter gene designed to govern the expression of a human insulin gene shows such tissue specific enhancement activity that the human insulin gene can be integrated to the chromosome of keratinocytes with the aid of Drosophila""s transposase, and insulin can be produced by the keratinocytes in a quantity sufficient to maintain normal blood glucose levels.
Therefore, it is an object of the present invention to provide a non-viral, recombinant insulin expression vector suitable for use in gene therapy for diabetes mellitus.
It is another object of the present invention to provide a non-viral vector containing a DNA sequence coding for Drosophila""s P-transposon, suitable for use in gene therapy.
It is a further object of the present invention to provide use of the non-viral, recombinant insulin expression vector in treating diabetes mellitus.
It is still a further object of the present invention to provide use of the non-viral vector containing a DNA sequence coding for Drosophila""s p-transposon in integrating genes to mammalian chromosomes.
It is still another object of the present invention to provide use of the non-viral vector containing a DNA sequence coding for Drosophila""s P-transposon in treating diabetes mellitus.
It is yet another object of the present invention to provide a method for treating diabetes mellitus by gene therapy.
It is yet a further object of the present invention to provide a composition for gene therapy for insulin-dependent diabetes mellitus, which is safe and easy to apply to humans.